The pathogenesis and aetiology of pre-eclampsia (PE) is still unclear. We investigated the role of angiogenic, anti-angiogenic and vasoactive factors in black South African women with early- and late-onset PE. Serum soluble fms-like tyrosine kinase 1 (sFlt-1), soluble vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) levels were determined using the ELISA technique, and placental mRNA expression levels of sFlt-1, VEGF, PlGF and AT1 receptors were determined using real-time PCR.Serum sFlt-1 levels were significantly elevated and PlGF significantly reduced in early-onset PE compared to the normotensive group. Placental VEGF mRNA expression levels were significantly reduced in the late-onset preeclamptic group compared with the normotensives. The placental mRNA expression of AT1 receptor in the late-onset pre-eclamptic group was relatively raised compared to the normotensives, suggesting hypersensitivity to pressor agents.We believe that the excess of serum sFlt-1 and reduced VEGF and PlGF levels favour an anti-angiogenic state and endothelial dysfunction leading to PE, and that the aetiology and pathogenesis of early- and late-onset PE differ. Pre-eclampsia (PE) is a pregnancy-specific syndrome that causes substantial maternal, foetal and neonatal morbidity and mortality worldwide.Despite years of research, the exact aetiology of PE remains unknown.It is believed that placental ischaemia during stage 1 may lead to placental production of a soluble factor or factors that cause maternal endothelial dysfunction.9When the maternal endothelium is deprived of these angiogenic factors (VEGF and PlGF) and in the presence of excess anti-angiogenic factors such as sFlt-1, it becomes dysfunctional and leads to the clinical syndrome of hypertension and proteinuria.13Most studies on angiogenic/anti-angiogenic factors, however, have been done in high-income countries. Furthermore, there may be racial variations in both the incidence of PE and the clinical features at presentation. For example, the incidence of PE is much higher in South Africa and it has a much more aggressive and rapid clinical course of presentation, leading to significant mortality.n = 30); chronic hypertensives (n = 9) ; early-onset pre-eclamptics (≤ 27 weeks gestation) (n = 10) and late-onset pre-eclamptics (≥ 28 weeks gestation) (n = 9).Following institutional ethical permission, pregnant women who gave written, informed consent were recruited from the labour ward of a regional hospital in the KwaZulu-Natal province, South Africa. Following enrolment, the participants were grouped as follows: clinically healthy normotensive controls (Definitions: pre-eclampsia was defined as new-onset hypertension (blood pressure 140/90 mmHg) and proteinuria (≥ 2+ on testape) after the 20th week of pregnancy. Chronic hypertension was defined as women with a history of hypertension in the non-pregnant state and who had hypertension at the first antenatal visit prior to 20 weeks’ gestation.All clinical data were recorded on a structured form and included the highest blood pressure measurement, level of proteinuria, maternal age, parity, gestational age, mode of delivery and neonatal outcomes.Pre-partum blood samples were obtained from participants who were in early labour. Postpartum central placental samples were obtained soon after delivery of the babies. The blood samples were centrifuged at 4°C at 3 000 rpm for 30 minutes and aliquots of serum samples and placental tissue samples were stored at –70°C until used. All participants with hypertension (chronic hypertensives and pre-eclamptics) were on antihypertensive medication, namely methyldopa.The quantitative sandwich enzyme immunoassay technique was performed on serum samples to analyse for the levels of sFlt-1, VEGF and PlGF using Quantikine ELISA kits . All assays were performed in duplicate according to the manufacturer’s instructions.RNA was extracted from placental samples using a protocol previously described,18p-value of < 0.05 was considered statistically significant. The Kruskal-Wallis test was done to assess for any overall significant differences across the four groups within each variable in clinical data, as well as between the groups for each mRNA gene expression. This was followed by the Mann-Whitney test to determine which groups displayed these differences.All values are expressed as means ± SEM (standard error of mean). Statistical tests were performed using SPSS version 15.0 . A p < 0.05).One-way ANOVA was performed to determine if there were significant differences in the serum concentrations of sFlt-1, PlGF and VEGF, and the mRNA expression levels of sFlt-1, VEGF, PlGF and the AT1 receptor among the groups. The Tukey-Kramer multiple comparison test and the Mann-Whitney test compared each group against the other and determined which two groups displayed a significant difference (p > 0.05).The body mass of the neonates in the early-onset (2.83 ± 0.38 kg) and late-onset (3.03 ± 0.16 kg) pre-eclamptic groups were lower than those of the normotensive (3.24 ± 0.08 kg) and chronic hypertensive (3.34 ± 0.17 kg) groups. However, these differences were not statistically significant (p < 0.05) The serum sFlt-1 concentrations in the normotensive controls (9 603 ± 1 797 pg/ml) were significantly less than those in the early-onset pre-eclamptic group (26 682 ± 5 482 pg/ml) (p = 0.001). Furthermore, there was a significant difference in the levels of PlGF between the normotensive (0.83 ± 0.11 pg/ml) and chronic hypertensive (0.42 ± 0.063 pg/ml) groups (p < 0.05) The serum concentrations of VEGF in all four groups were below the detectable limit of the assay. The relative placental mRNA expression levels of sFlt-1, VEGF, PlGF and AT1 were compared across the four groups and were normalised to the housekeeping gene, GAPDH. Data are expressed as fold changes.The highest mRNA expression level of sFlt-1 was found in the late-onset pre-eclamptic group (1.789 ± 0.513 fold) and the lowest level was found in the chronic hypertensive group (1.009 ± 0.162 fold) p < 0.05). The VEGF mRNA expression level in the normotensive group compared with the early-onset pre-eclamptic group (0.943 ± 0.28) had a p-value very close to being significant (p = 0.051).The highest level of VEGF mRNA expression was found in the normotensive group (2.261 ± 0.42 fold) p = 0.055). The ratio in the late-onset pre-eclamptic group and chronic hypertensive group were 2.17- and 2.34-fold greater, respectively than the normotensive group.As shown in Although the differences in mRNA expression levels of PlGF among the four groups were not statistically significant, the late-onset pre-eclamptic group had the lowest level (0.81 ± 0.34) while the chronic hypertensive group had the highest level (2.17 ± 0.94) and the latter was 1.80-fold higher than in the normotensive group (1.205 ± 0.4818) p-value very close to significance (p = 0.0592 fold). In addition, a positive correlation was found between the AT1 receptor concentration and diastolic blood pressure in the late-onset pre-eclamptic group (p < 0.05).Among all four groups, the late-onset pre-eclamptic group (23.20 ± 13.36 fold) had the highest level of AT1 mRNA expression In this study healthy, normotensive, pregnant women served as an appropriate control by representing the physiological changes that occur during uncomplicated pregnancy, which was a point of reference for the pathological conditions of chronic hypertensive pregnancy and PE. Chronic hypertensive pregnancy was a significant reference for the pre-eclamptic groups, indicating that any observations that were common to these groups may be attributed to the high blood pressure that was a common characteristic. Therefore if an observation was made in the pre-eclamptic groups but not the chronic hypertensive group, it could possibly be attributed to the disease pre-eclampsia.The lower gestational ages in the early- and late-onset pre-eclamptic groups in comparison with the normotensive group may have been as a result of early delivery preventing further complications, as the only known cure for the syndrome is delivery of the baby and placenta.As expected, the systolic and diastolic blood pressure measurements in the chronic hypertensive and pre-eclamptic groups were significantly higher than in the normotensive controls. Interestingly, the systolic and diastolic blood pressures in the late-onset pre-eclamptic group were significantly higher than in the chronic hypertensive group, indicating that the hypertension in late-onset PE may be more severe than that of chronic hypertension. This may be attributed to the hypertension-inducing effects of sFlt-1, as previously shown in the murine model.19Recent reports suggest that hypertension in response to sFlt-1 may be associated with increased circulating vascular superoxide concentrations and that reactive oxygen species could be involved in mediating the blood pressure response to excessive sFlt-1 during pregnancy. It has been suggested that higher serum sFlt-1 concentrations could play a role in endothelial dysfunction.10The serum VEGF levels in all our groups were below the detectable limit of the assay, which is to be expected in the antenatal periodp = 0.051), compared with the normotensive group. These findings could also have been contributing to the VEGF deficiency in the serum of the pre-eclamptic women. Our study therefore concurs with previous reports showing evidence of reduced VEGF expression in the placentae from pregnancies complicated by PE.26Interestingly, the highest level of VEGF mRNA expression was found in the normotensive group. The level of VEGF mRNA expression was significantly lower in the late-onset pre-eclamptic group, and very close to being significantly lower in the early-onset pre-eclamptic group , suggesting that the RAS may be implicated in the pathogenesis of PE in this group. This may also indicate that late-onset PE is a different disease from early-onset PE and, therefore may actually be a disease that has a closer association with the RAS than the angiogenic/anti-angiogenic theory, as is the case in early-onset PE.In this study the AT1 receptor expression levels were relatively lower in the normotensive, chronic hypertensive and early-onset pre-eclamptic groups than in the late-onset pre-eclamptic group. This can be expected in the normotensive group since in normal pregnancies there is a reduced response to vasopressors.p < 0.05). Unfortunately, the factors that activate the AT1 receptors, including angiotensin II and the AT1 auto-antibodies were not quantified in this study.The raised AT1 receptor mRNA expression level in late-onset PE may be due to raised AT1 autoantibodies in this group.Further studies using a larger sample size and measuring AT1 receptor mRNA expressions, AT1 autoantibodies and blood pressure in these two groups are necessary to prove this hypothesis. Interestingly, a recent study showed that the increase in AT1 autoantibodies might represent a better marker for late-onset PE, whereas sFlt-1 is a better marker for early-onset PE.50To the best of our knowledge, this is the first time it has been shown in black South African women that placental AT1 gene expression levels were relatively elevated in late-onset PE compared with the early-onset pre-eclamptic, normotensive and chronic hypertensive groups. In another study conducted on black South African women, it was shown that the common RAS polymorphisms could not be used to predict PE.51It has previously been shown that the blunted response to angiotensin II during normal pregnancy is lost in pre-eclamptic patients, who show a hypersensitivity to vasopressors.53Most studies on PE have been conducted on Caucasian participants, and there is a paucity of information on other ethnic groups. It is well documented that socio-demographic factors such as race have an influence on the incidence of PE in a population.We recommend that future research should also incorporate the investigation of the heterodimerisation of the AT1 and bradykinin B2 receptors in black South African women. Furthermore, the interplay between the angiogenic/anti-angiogenic pathway, RAS and other theories, such as that of oxidative stress and the role of the inflammatory system in PE, should be explored.Two of the limitations of this study were the small sample size that affected the statistical analysis, and the fact we did not measure serum levels of angiotensin II and AT1 autoantibodies.The findings of this study lead us to conclude that the aetiology and pathogenesis of early- and late-onset PE are, to some extent different, especially in our population sample. Our data demonstrate that while a predominance of an anti-angiogenic state may be an important role player in both early- and lateonset PE, in the latter, AT1 receptor up-regulation and the RAS could also play a role.


Objective. The dosage, duration, and the benefits of high-dose steroid treatment and outcome in biliary atresia (BA) remain controversial. In this study, we evaluated the impact of high-dose steroid therapy on the outcome of BA after the Kasai procedure. Methods. Intravenous prednisolone administration was started 1 week after surgery, followed by 8 to 12 weeks of oral prednisolone. Total bilirubin (TB) levels , early onset of cholangitis, and two-year native liver survival were evaluated. Results. 53.4%, 56.9%, and 58.1% of the patients in the high-dose steroid group were jaundice-free 3, 6, and 12 months after surgery, respectively; these values were significantly higher than the 38.7%, 39.4%, and 43.3% of the low-dose steroid group. One year after surgery, the incidence of cholangitis in the high-dose group (32.0%) was lower than that in the low-dose group (48.0%). Infants with native liver in the high-dose group had a better two-year survival compared to those in the low-dose steroid group (53.7% versus 42.6%). Conclusions. The high-dose steroid protocol can reduce the incidence of cholangitis, increase the jaundice-free rate, and improve two-year survival with native liver after the Kasai operation. Biliary atresia (BA) is a progressive fibroobliterative disease of the biliary tract occurring in infants . The surSince 2007, our institution had started to treat BA patients who received the Kasai operation with a high-dose steroid protocol. We also used antibiotics and ursodeoxycholic acid to prevent cholangitis and stimulate bile flow as well as bile drainage –12. To en = 380) who underwent the Kasai operation for treatment of BA from January 2004 to December 2009 in the Children's Hospital of Fudan University. All study protocols were approved by the Institutional Review Board at the Children's Hospital of Fudan University, IRB 2010167. The patient groups and treatments were designed as shown in A retrospective study of prospectively collected data was performed on all children (μmol/L). This high-dose steroid protocol also included intravenous antibiotics  for 4 weeks, followed by 8 to 12 weeks of oral antibiotics . This protocol also included oral ursodeoxycholic acid (20 mg/kg/d) for 12 to 24 months.Of the 380 treated patients, 127 children (from January 2004 to December 2006) received a low-dose steroid protocol and 253 children (from January 2007 to December 2009) were treated with a uniform postoperative protocol of high-dose steroids, long-term intravenous antibiotics, and ursodeoxycholic acid. In the groups receiving high-doses of steroids, intravenous prednisolone  was started one week after surgery, followed by 8 to 12 weeks of oral prednisolone, which started at 4 mg/kg every other day and was gradually tapered until patients became jaundice-free  were used for 1 week after surgery. Children in this group also received oral ursodeoxycholic acid (20 mg/kg/d) for 12 to 24 months.t-test. All data were reported as mean ± SD. A value of P < 0.05 was considered significant.Statistical analysis was performed with SPSS 13.0 for Windows. The Pearson's chi-square test or Fisher's exact test was used to compare qualitative variables. Quantitative variables were analyzed by the Student's During the study period, 380  children underwent the Kasai procedure. Among them, no family history was found and 54 (14.2%) children had at least one abnormality . The average birth weight in the low-steroid (127) and high-steroid (253) groups was 3390 ± 438 g and 3420 ± 397 g, respectively. Twelve children in the low-dose steroid group and 7 in the high-dose group were lost to follow-up after 12 months and were included in the analysis of the postoperative bilirubin level and incidence of cholangitis but not in the analysis of the survival with native liver rates.P = 0.032). Among the children (361) who underwent the two-year follow-up, the survival with native liver rate of children who received the Kasai operation at an age of <60 days was 55.1% (43/78). The survival with native liver rate dropped slightly in infants who received the Kasai operation between 60 and 90 days  and above 90 days . However, no significant difference was found among the different age groups (P > 0.05) (The average age at surgery in the low-steroid group (74 ± 31 days) was higher than that in the high-steroid group  .μmol/L) compared with those in the low-steroid group . At 6 and 12 months after surgery, the number of children with normal bilirubin also showed significant differences between the two groups  and , respectively. According to the diagnosis standard, 12 months after surgery, 48.0% (61/127) of children in the low-dose steroid group suffered from cholangitis but only 32.0% (81/253) of those in the high-dose steroid group had cholangitis. The two-year survival with native liver rate in the high-dose steroid group was 53.3% (131/246). The survival with native liver rate dropped markedly in children who received low-dose steroids . There was a significant difference between the two groups (P = 0.001) (P > 0.05)  had normal bilirubin  . However > 0.05) . Hemorrh+-K+ ATPase, which increases canalicular electrolyte transport and stimulates bile flow independent of bile salt concentration [Begun as a desperate attempt to treat refractory ascending cholangitis in infants with BA following the Kasai operation, the use of steroids has evolved into a commonly used post-HPE therapy believed to improve clinical outcomes in BA. Karrer and Lilly  proposedntration . In addintration .The clinical use of steroids in BA has increased dramatically in recent years following the publication of a series of reports demonstrating better outcomes with routine post-HPE steroid therapy compared to a variety of historical or concurrent control groups –7. MurajIn the current study, the low-dose steroid group received the same protocol as the patients studied by Muraji and Higashimoto . HoweverIn our study, the two-year survival with native liver rate in the two groups (53.3% and 38.3%) is lower than that observed in the three studies mentioned above . However, those studies included lower numbers of children, making it difficult to compare survival with native liver rates between these previous reports and the current study. The survival with native liver rate in the high-dose steroid group in the current study (53.3%) is similar to that reported in a nationwide survey in Japan in 2004 (a group of 108 children received steroids at doses >4 mg/kg/d) .One of the limitations of this study is retrospective collection of data, which is inferior to prospective randomized study. Although all surgeries were performed by a single physician with the same surgical technique and a larger number of patients were included in this study, whether the high-dose steroid protocol is primarily responsible for the improved outcome remains elusive, given that our current study has not been randomized or rigorously controlled for a host of potential confounding variables, and it is not empowered to definitively answer the question of causality. For instance, only the high-dose steroid group received long-term intravenous and oral antibiotics to lower the incidence of cholangitis. In the high-dose steroid group, the age at the time of surgery was slightly younger than that of the low-dose steroid group. Although some studies have concluded that children who received the Kasai procedure earlier always have a better prognosis, the age at time of surgery had no significant influence on survival with native liver in the present study –21.Miyano and colleagues  showed t


The application of chemotherapeutic drug adriamycin (ADR) in cancer therapy is limited byits side effects like high toxicity and insolubility. Nanomedicine offers new hope forovercoming the shortcomings. But how to increase in vivo stability and to control intracellulardrug release is a key issue for nano-based formulations. Herein, the hydrophobic ADR wassuccessfully linked to the biocompatible human serum albumin (HSA) by disulfide bond3-(2-pyridyldithio) propionyl hydrazide (PDPH), resulting in amphiphilic HSA-ADR. Thenovel ADR-HSA micellar NPs which were thus assembled exhibited a well-defined stablecore shell structure with glutathione (GSH) sensitive linkers. The stable PDPH linkers atextracellular level were broken by GSH at intracellular level with a controlled ADR releaseprofile. The in vitro cytotoxicity against gastric cancer cells (NCI-N87) was obviouslyenhanced by such redox-sensitive ADR-HSA NPs. Additionally, as observed by IVISLumina II Imaging System (Xenogen), the intratumor accumulation of ADR-HSA NPs wasmuch higher than that of HSA/ADR NPs due to its high stability. Consequently, the in vivotumor inhibition was significantly promoted after intravenous administration to the Balb/c nude micebearing gastric tumors. These in vitro/vivo results indicated that disulfide-bond-containingADR-HSA NPs were an effective nanodrug delivery system for cancer therapy. Adriamycin (ADR) is a kind of anthracyclines antitumor drug, which has strong cytotoxic effect and has been widely used in the treatment of liver cancer, lymphoma, gastric cancer, and breast cancer , 2. But The HSA based formulation that was the paclitaxel (PTX) albumin nanoparticles solution (ABRAXANE) developed by American bioscience has been approved by the Food and Drug Administration (FDA) for clinical cancer therapy . Clinicaμmol/L) [It is known that the intracellular environment is different from extracellular environment which was characterized by the lower pH (4–6) and the redox states . Intraceμmol/L) . The GSHμmol/L) , 18. In μmol/L) , 20. Allμmol/L) –23. Amonμmol/L) –28. At pμmol/L) .In this study, the disulfide bond PDPH was utilized to link the ADR to the HSA, resulting in reduction-sensitive amphiphilic copolymer HSA-ADR. Then the novel micellar-like ADR-HSA nanoparticles were assembled by a rotary evaporation technique. The nanocarrier's properties, in vitro and in vivo antitumor effects were systemically investigated. This study provided an easy and feasible idea for the design and preparation of reduction-sensitive nanodrug delivery system.Adriamycin was obtained from Dalian Meilun Biology Technology Co. 3-(2-Pyridyldithio) propionyl hydrazide (PDPH) was obtained from Pierce. HSA was obtained from Sigma with purity of 99%. 2-Iminothiolane hydrochloride (2IT) was purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM) and Fetal bovine serum (FBS) were purchased from Gibco Co., USA. All chemicals were in analytic reagent grades.2.The human stomach cancer NCI-N87 cell line, which was purchased from Shanghai Institute of Cell Biology, was cultured in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin at 37°C with 5% COMale Balb/c mice  were obtained from Shanghai Experimental Animal Center of Chinese Academic of Sciences  and kept under SPF conditions. The animal experiments were performed in accordance with guidelines provided by the ethics committee of the Second Military Medical University, Shanghai, China.ADR-HSA was synthesized according to the procedure shown in 2 atmosphere to form a thin film. Then the flask was connected with the equipment of rotation evaporation. The ADR-HSA film was eluted by 2 mL dd water; as a result, ADR/HSA NPs solution with concentration about 0.25 mg/mL was obtained. On the other hand, the ADR/HSA NPs solution were just prepared by mixing the drug ADR with HSA with the same concentration as ADR-HSA and dissolved in 2 mL dd water. For evaluation of cytotoxicity of the ADR-HSA nanoparticles (ADR-HSA NPs), the blend of the ADR/HSA nanoparticles (ADR/HSA NPs) at the same ADR concentration was served as control.In the experiments, 0.5 mg ADR-HSA was dissolved in 1.5 mL chloroform followed by drying under Nζ) of ADR/HSA and ADR-HSA NPs was tested by a Malvern Zetasizer 3000HS  using 5 mM NaCl as the baseline.The size distribution of ADR-HSA NPs was tested by the dynamic laser light scattering instrument  at the scattering angle of 30°. The surface potential , PL-RB solution (0.5 mg/mL) was dropped on a 200-mesh Formvar-free carbon-coated copper grid (Ted Pella Type-A). After water evaporated, the sample was inversely covered on a small drop of 2% hydrodated phosphotungstate (PTA) solution. The conventional TEM images were obtained at 100 kV.The in vitro drug release was conducted by the dialysis method. The ADR-HSA NPs solution in the membrane with concentration of 0.5 mg/mL was dialysed against 500 mL PBS at the pH ~7.3. At the predesigned time, about 0.05 mL ADR-HSA NPs solution was sampled and diluted by PBS to a volume of ~1 mL. At the same time, about 0.05 mL PBS was added to the ADR-HSA NPs solution inside membrane. For evaluating the effects of PDPH spacer on drug release, different concentrations of glutathione (GSH) were added to the drug solution. The ADR-HSA NPs solutions sampled at different time points were tested by the florescent at wavelength ~480 nm. The drug concentrations outside-/inside-membrane were calculated and the release profile was converted.μL per hole. The petri dish was then kept in a 5% CO2 incubator at 37°C overnight. The next day, the ADR/HSA NPs and ADR-HSA NPs suspension were diluted with DMEM containing 10% FBS (the concentration of ADR was 1 μM). The culture medium was sucked out of the dish; then the sample solutions were added and incubated for 2 h at 37°C. The sample solution was removed, and the cells were washed using PBS solution twice and observed under laser scanning confocal microscopy .The NCI-N87 cells in logarithmic growth phase were digested by 0.25% trypsin (containing 0.05% EDTA). The cells were resuspended in DMEM containing 10% FBS and inoculated on a glass bottomed petri dish, with 150 Asample, Ablank, and Acontrol are UV absorption at 485 nm from cell incubated with samples, the culture medium and the cell without samples.The CCK-8 method was used to determine the in vitro growth inhibition effect of ADR/HSA NPs and ADR-HSA NPs to tumor cells. The NCI-N87 cells were digested using 0.25% trypsin, inoculated in 96-well plates  with 2000 cells per hole, and cultured for 12 h. The samples of ADR/HSA NPs and ADR-HSA NPs were diluted to 6.25 nM to 200 nM (ADR). The culture medium in 96-well plates was removed and the different concentrations of samples were added and incubated for 12 h. The sample solutions were removed, and the CCK-8 solution was added. After 10 min, the absorbance was detected at 490 nm by enzyme-labeling instrument . The percentage of surviving cells was calculated according to the following equation :(1)Survi6 NCI-N87 cells (in 100 μL culture medium) to develop xenografts tumor. After about 2 weeks, the volume of tumors reached about 50 mm3. For the in vivo distribution, mice bearing NCI-N87 tumor were randomly assigned to 3 groups with 3 mice/group . Loading fluorescent isothiocyanate (FITC) HSA/ADR and HSA-ADR NPs with equivalent to 5 mg/kg dosage were administered via tail vein. 24 hours later, the tumors were viewed by IVIS Lumina II Imaging System (Xenogen), which was taken to capture the visible light photograph and luminescent image. Noted here, the mice with FITC administration at 0 hour were used as control. The in vivo images were observed with IVIS imaging system (excitation 500 nm) and recorded by a built-in CCD camera [2/N2. Mice were sacrificed after 24 h and the heart, liver, kidney, tumor, and spleen were excised. These organs were also imaged with the same excitation wavelength. Then the organs were collected and immediately fixed in formalin for 1 h. The organs were frozen in tissue-Tek-OCT and cryosections. Frozen sections were cut at 10 μm and fixed with acetone at −20°C. After washing with PBS, sections were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride  and visualized by the confocal microscopy.Firstly, the Balb/c nude mice were inoculated subcutaneously on the right back with 5 × 10D camera . For fur6 cells in 200 μL DMEM medium) subcutaneously. The nude mice were used for the experiment when the tumor tissue reached to 60 mm3. Three groups of nude mice models (n = 5) were intravenously injected with PBS (N.S.), ADR/HSA NPs, and ADR-HSA NPs, successively for 3 days. The dosage of ADR for each injection was 5 mg/kg, and the total dose of ADR for each group was 300 mg/mice. The volume of tumor tissue was measured every 2 days from the 1st day to the 33rd day. The tumor volume (V) was calculated according to the following equation [L and W were the longest and shortest diameters. The related tumor volume was calculated according to the formula: related tumor volume = Mean tumor volume at day recorded/mean tumor volume at day 0.The nude mice model was constructed by injection of NCI-N87 cells (1 × 10equation : (2)Vtumt-test was performed to measure the statistical differences of different groups . The IC50 value was calculated by using the Prism ver5.02 program.Student's The chemical synthesis of protein-drug conjugates was shown in μM as summarized in The in vitro cytotoxicity of ADR, ADR/HSA NPs, and ADR-HSA NPs against NCI-N87 tumor cells was shown in p < 0.01) and ADR-HSA NPs (p < 0.001) showed a significantly more enhanced antitumor effect. Furthermore, the antitumor effect of ADR-HSA NPs was significantly higher than that of ADR/HSA NPs (p < 0.001). The tumor inhibitory rate of ADR/HSA NPs and ADR-HSA NPs was 22.91% and 69.98%, respectively. The enhanced inhibitory effect of ADR-HSA NPs should be in accord with their reduction sensitivity and the increased cellular uptake by tumor cells.The results of in vivo antitumor effects of ADR/HSA NPs and ADR-HSA NPs were shown in It was known that the clinical application of small molecular chemotherapeutic drug adriamycin (ADR) was limited by its low solubility and high side effects. For overcoming the drawbacks, new nanoformulations based on liposomes, micelles were developed, among which, a successful sample was the HSA/PTX blend. This nanomedicine has been widely used in clinics. However, there still existed serious cytotoxicity such as bone marrow suppression, immunosuppression, and cardiac toxicity, which still strongly limited its clinic application. The limitation was attributed to the in vivo instability, drug leakage, and insufficient drug release. In this paper, we adopted a simple and feasible rotary evaporation and hydration technique to prepare novel reduction-sensitive nanoparticles ADR-HSA NPs. As evaluated by the UV and DLLS, the number of ADR conjugated on one HAS was about 15–20. As shown in In this new ADR-HSA NPs formulation, the outer shell was a biocompatible protein HSA. The inner core was composite of the hydrophobic drug ADR, while in the middle of the nanoparticles was a redox-sensitive linker PDPH, which can be degraded at a related high concentration of GSA, leading to the controlled drug release as shown in μmol/L. The PDPH linkers were thus remarkably degraded by such a high concentration of GSH, which resulted in the ADR release. Therefore, disulfide-contained drug carriers can timely release drugs dependent on the concentration of GSH in tumor cells. So compared with the HSA/ADR system, the ADR-HSA NPs significantly enhanced tumor growth inhibition as shown in The above-mentioned serum stability of the ADR-HSA NPs further enhanced the in vivo tumor accumulation as shown in In summary, by a facile chemical conjugation of chemotherapeutic drug ADR to the biocompatible protein HSA, an amphiphilic molecule HSA-ADR was obtained with disulfide bond PDPH. Then the novel micellar-like ADR-HSA NPs were constructed by rotary evaporation method, which exhibited a well-defined core shell structure and GSH-sensitive releasing profile. The ADR release was controlled by the concentration of GSH. The in vitro cytotoxicity against NCI-N87 gastric cancer showed that such ADR-HSA NPs containing redox-sensitive PDPH linkers obviously enhanced the cellular uptake of ADR. In addition, the intratumor accumulation of such ADR-HSA NPs was much higher than that of the HSA/ADR blend due to its high serum stability and tumor accumulation. Consequently, the in vivo tumor inhibition by this ADR-HSA NPs was significantly promoted as intravenous administration to the Balb/c nude mice bearing NCI-N87 gastric tumor. These results indicated that disulfide-bond-containing ADR-HSA NPs were an effective nanodrug delivery system for targeting therapy of gastric cancer. This study may provide a new idea for the development of nanomedicine.


The Minimal Eating Observation and Nutrition form – version II (MEONF – II) is a recently developed nursing nutritional screening tool. However, its inter- and intrarater reliability has not been assessed.Inpatients  were assessed by eight nurses  using the MEONF-II on two consecutive days .Six patients were at moderate/high undernutrition risk. Inter- and intrarater reliabilities (Gwet’s agreement coefficient) for the MEONF-II 2-category classification (no/low risk versus moderate/high risk) were 0.93 and 0.81; for the 3-category classification (no/low – moderate – high risk) reliabilities (Gwet’s weighted agreement coefficient) were 0.98 and 0.88; and total score inter- and intrarater reliabilities (intraclass correlation) were 0.92 and 0.84.Reliability of MEONF-II nurse assessments among adult hospital inpatients was supported and the tool can be used in research and clinical practice. The Minimal Eating Observation and Nutrition form version II (MEONF-II)-3 was deThe MEONF-II  in four general adult hospital wards  were eligible for inclusion. Fifty-six (76%) of 74 available inpatients gave informed consent to participate, of which every second (n = 28) was included in the study of inter- and intrarater reliability. The other patients were included for the purpose of estimating the pointprevalence of undernutrition risk at the hospital (not presented here). Four patients were not available for intrarater reliability assessment on day two and were thus excluded. The final sample was 24 patients. For test-retest reliability studies a minimal sample size of 15 to 20 suffice,14.Two registered nurses at each of the four participating wards (n = 8) performed the data collection. Before commencing data collection, they received brief training (45–60 minutes), including pre-testing of the forms.Data were collected in 2012. Two nurses on each ward assessed the patients according to the MEONF-II, in parallel with each other . The nurses did not communicate during the assessment or about their findings afterwards. The nurses in each pair agreed upon who should be designated to be the "first" and "second nurse" (for the purpose of interrater reliability). During day 2 the MEONF-II assessment was repeated, by the same nurse who made the assessment of the same patient on day 1, to assess the intrarater reliability.MEONF-II height and weight measurements were conducted using standard equipment available at the included units, and the patients were observed while eating and asked about eating difficulties and unintentional weight loss. Data collection was made under conditions as close as possible to clinical daily routine.The data collection forms, including the MEONF-II and the manual were translated into Icelandic and back translated into Swedish in collaboration with a professional translator.In addition to the MEONF-II, dependence in activities of daily living (ADL) was assessed using a modified Katz ADL-index,15. It sIn addition, three single-items regarding fatigue/tiredness, depression and perceived health were applied. The fatigue/tiredness and depression items asked whether respondents had gotten tired without any specific reason and felt gloomy and depressed, respectively, today/during the last days ,16,17. TU test depending on level of data. Inter- and intrarater reliability was analysed with proportion exact agreement (PA), Kappa statistics (K), quadratic weighted K (Kw), Gwet’s agreement coefficient (AC1), weighted AC1 (AC1w), and intraclass correlation coefficient (ICC) and their 95% Confidence Intervals (95% CIs)[K and AC1 for the 2-category classification (identifying no/low risk versus moderate/high risk), Kw and AC1w for the 3-category classification (no/low – moderate – high risk), and ICC for the total score[Comparisons were made between those included in the study and those not using Chi-square and Mann–Whitney 95% CIs)-22. Sincal score.K when there are low frequencies in some cells. The advantage of K is that it accounts for both PA and the chance agreement, and Kw considers partial agreement[K statistics can sometimes be low despite high levels of agreement[K we calculated the maximum obtainable kappa (Kmax), and the prevalence-adjusted bias-adjusted kappa (PABAK). If the prevalence index is high, i.e. the prevalence of a positive rating is very high or low, chance agreement is also high and K is reduced accordingly. A bias index close to zero indicates that the disagreement between raters is close to symmetrical. The higher the bias index is, the higher the K will be. Thus, PABAK adjusts for both prevalence and bias. Kmax shows the maximum obtainable kappa for the set of data used[K statistics and has therefore been recommended as an alternative or complement to K[K and AC1 values below 0.20 are regarded as poor, 0.21-0.40 as fair, 0.41-0.60 as moderate, 0.61-0.80 as substantial and >0.80 as almost perfect agreement[PA does not account for chance agreement but is useful as a complement to other statistics, particularly to greement. Howevergreement. In ordeata used. The AC1ment to K. K and Agreement,26.Regarding the ICC we used a two-way mixed model . ICC valThe ethical committee of Akureyri Hospital (case 2/2012) approved the study and the use of personal data from the study was notified to the Data Protection Authority (S5594/2012). The study conforms to the provisions of the Declaration of Helsinki in 1995 (as revised in Edinburg 2000) including informed consent and patient anonymity.The eight nurses  had a median age of 51 years , had been working as nurses for a median of 16 years (7–38 years), and in the current hospital for a median of 12 years (4–32 years). The nurses were general hospital ward nurses and none of them had prior experience with the MEONF-II.There were no significant differences between patients included in the study and those not Table .The prevalence of moderate/high undernutrition risk on day 1 was 21-25%. On day 2 the prevalence was 21% (Table K/AC1) for the MEONF-II 2-category classification (no/low risk versus moderate/high risk) were ≥0.81 except for intrarater K (0.65). Similarly, for the 3-category classification (no/low – moderate – high risk) inter- and intrarater reliabilities (PA/Kw/AC1w) were were ≥0.83 except for intrarater Kw (0.62). For the total MEONF-II scores ICC values were above 0.80 for both inter- and intrarater reliabilities  of 21 or 25%. This is similar to previously reported rates according to the MEONF-II. In an Icelandic study the baseK 0.53) is similar to that found for the Mini Nutritional Assessment total score  between two geriatric clinicians independently assessing 39 in-hospital patients[K varied between 0.34 and 0.88, and was highest with experienced assessors[Kw) for the 3-category classifications[MEONF-II assessments were found to exhibit generally good reliabilities following a relatively low amount of training. The interrater reliability was particularly high and comparable to the figures found for other nutritional screening tools. The interrater reliability for MEONF-II total score , AC1 (0.81) and PA (0.88). Indeed, when K is low despite high PA it has been suggested to instead rely on the more robust AC1[Arguably, the most important reliability is that of the 2-category classification of MEONF-II scores as it initially might be used for determining if a patient shall receive a nutritional intervention and/or a more thorough assessment. This reliability was found to be high  between raters but lower  within raters . This may be due to several possible explanations. There might have been a change in nutritional risk between the two time points, due to improvement or decline in health status. Differences in total scores in assessments between the 24 hours affected the nutritional risk classifications of four patients; two in each direction, i.e. towards better and worse nutritional risk status, respectively. The choice of time period between assessments is primarily a balance between the risk for changes in the underlying trait and recall effects. A seconK, whereasK. The difbust AC1. Our obsAccording to Kottner et al. 12), reliability values of 0.60-0.80 may be regarded as sufficient for group-level applications, whereas values of at least 0.90 are required for individual patient assessments, when important decisions are based on the assessment[, reliabiK statistics, despite high proportions of exact agreement, is likely to represent a methodological artefact. The MEONF-II can be used in a reliable way in research and clinical practice.This study provides support for the inter- and intrarater reliability of MEONF-II nurse assessments among adult hospital inpatients. Somewhat compromised intrarater reliability according to The authors declare that they have no competing interests.All three authors have substantially contributed to the study. AW conceived and designed the study, carried out the analyses and drafted the manuscript. ÓT coordinated the translation process and the data collection. PH and ÓT revised the manuscript and interpreted the data. All three authors gave final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6955/13/18/prepubMEONF-II .Click here for file


The aim of the current study was to compare levels of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in lupus patients and controls and to investigate their association with clinical phenotype, disease activity and damage.We compared levels of serum VCAM-1 and E-selectin in 178 female lupus patients and 69 age-and sex-matched controls. Using linear regression we also examined the association between these markers and disease activity, damage, renal and skin involvement as well as clinical and subclinical cardiovascular disease.p < 0.001). E-selectin was also associated with overall damage and carotid plaque : 0.27  and 0.26 ). Whilst there was no significant difference in VCAM-1 levels between groups overall, we found a significant association between VCAM-1 and with active renal disease : 1.10 ).E-selectin was increased in patients compared to controls (median (IQR) 10.5  vs 7.86  ng/ml; E-selectin may act as a marker of cardiovascular risk in SLE, whilst VCAM-1 may have a role as a non-invasive biomarker for lupus nephritis activity. The cellular adhesion molecules (CAMs), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, are expressed exclusively on the surface of endothelial cells. Their function is to facilitate leucocyte-endothelial cell interactions and the transmigration of inflammatory cells to sites of inflammation. Their expression is upregulated by pro-inflammatory cytokines such as tumour necrosis factor (TNF)-alpha. These molecules are also shed into the circulation and may act as markers of endothelial activation and dysfunction.2 Some, but not all studies, have also found correlations with disease activity and particular disease phenotypes.4 For example, VCAM-1 appears to be of particular interest in renal disease, as raised levels have been found in active nephritis.6 In contrast, E-selectin levels have been less consistently associated with disease activity. However, one study did find increased E-selectin levels in patients with cutaneous manifestations.7VCAM-1 and E-selectin have been found to be elevated in patients with systemic lupus erythematosus (SLE).8 In SLE, where cardiovascular disease (CVD) is a leading cause of death,9 Gustafsson et al. found that high circulating levels of VCAM-1 were associated with cardiovascular mortality over a 12-year follow-up in an SLE cohort. Other studies have found associations with E-selectin and markers of subclinical CVD but results have been inconsistent. Such variation in results may reflect small sample sizes and heterogeneity of populations studied.In the general population, levels of CAMs have also been found to predict cardiovascular events and have been associated with hypertension.The aim of this study was to compare levels of VCAM-1 and E-selectin in a large cohort of lupus patients and a control population. We also investigated their association with lupus phenotype, in particular renal disease, skin disease and CVD as well as overall SLE disease activity and damage.10 Exclusion criteria included pregnancy and/or lactation within the past three months, recent infection within the past month and malignancy within the last 12 months.We carried out a cross-sectional study of adult female SLE patients and age- and sex-matched controls between August 2007 and July 2009. Ethical approval was obtained from the North West Regional Ethics Committee and informed consent was given by all participants. Patients were recruited from outpatient clinics in the North West of England. Controls were recruited via a “best friend” system, whereby patients invite a friend to participate in the study. All patients met four or more of the 1997 American College of Rheumatology (ACR) revised classification criteria for SLE.11 Active muco-cutaneous disease and active nephritis were defined as being present in patients scoring on either organ system within the relevant SLEDAI-2 K domains. A diagnosis of lupus nephritis ‘ever’ was recorded in patients with biopsy-proven disease or in patients with a history of haematuria and/or proteinuria and/or renal impairment who had a clinical diagnosis of nephritis in the absence of a formal biopsy. Damage was scored using the Systemic Lupus International Collaborating Clinics (SLICC) Damage Index (SDI).12 Fasting blood samples were drawn and, after centrifugation (1500 g for 15 minutes), plasma was stored at –80℃. Levels of E-selectin and VCAM-1 were measured using Duoset enzyme-linked immunosorbent assay (ELISA) development assays from R&D Systems . The E-selectin assay had a working range up to 6 ng/ml with a minimum detection limit of 0.1 ng/ml. The intra-assay coefficient of variation (CV) was 6% and the inter-assay CV was 7%. The VCAM assay had a working range up to 1 ng/ml with a minimum detection limit of 0.04 ng/ml. The intra-assay CV was 3% and the inter-assay CV was 8%.All subjects underwent a clinical assessment including history of demographics, lifestyle factors and medical history, and where necessary a review of the medical notes was carried out. The clinical phenotype and distribution of SLE manifestations were recorded. Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000 (SLEDAI-2K) score.13 In brief, three sets of IMT measurements were taken 1  cm from the carotid bulb bilaterally. From these, a mean IMT was calculated. Carotid plaque was defined as presence of two or more of the following parameters: IMT >1.5 mm, wall protrusion of >50% or increased wall echogenicity.Carotid ultrasound was performed, specifically evaluating carotid intima-medial thickness (IMT) and presence of carotid plaque. Measurements were taken using previously validated methodology.U test and Kruskal-Wallis test) were used to compare variables in patients and control subjects. VCAM-1 and E-selectin levels were log-transformed to satisfy the assumption of normality for linear regression. Linear regression models were employed to evaluate associations of levels of VCAM-1 and E-selectin with specific lupus features, SLEDAI-2K, SDI, and the presence of clinical and subclinical CVD. Significance level was set at p < 0.05. Our multivariable models included variables with a p value <0.2 on univariate analysis.Statistical analysis was performed using STATA11 software . Non-parametric tests (Mann-Whitney p = 0.066) (p = 0.004).We studied 178 patients and 69 controls with a median (interquartile range (IQR)) age of 53  and 50  years old, respectively (= 0.066) . OverallThe cohort included 160 (91%) Caucasians. The median (IQR) disease duration and SLEDAI-2K scores were 13  years, and 2  respectively. In total, 53 (30%) patients had a SLEDAI-2K >3. The median (IQR) SDI was 1  with 110 (61.8%) having at least one item of damage in the SDI. Five patients had active nephritis as defined using the SLEDAI-2 K score. All five patients had biopsy-proven lupus nephritis (class III-V) .Carotid plaque was observed in 78 (43.8%) patients on ultrasound and the median (IQR) IMT for the population was 0.063  cm. A greater number of SLE patients were on anti-hypertensive agents and statins than the control population . VCAM-1 levels did not significantly differ between groups (The median (IQR) E-selectin levels were significantly higher in patients compared to controls  vs 7.9  ng/ml, respectively; n groups .p < 0.001). In an age-adjusted linear regression model, VCAM-1 was significantly associated with active nephritis and inversely associated with smoking ): 0.006 ), therefore analysis was age-adjusted. The median (IQR) VCAM-1 levels were also significantly higher in patients with active nephritis than those with inactive nephritis and those with no history of nephritis  vs 276.7  vs 272.3  ng/ml, respectively;  smoking . These aIn an age-adjusted linear regression model, E-selectin was associated with carotid plaque and the SDI but not with overall disease activity or other SLE features . In a mu4 Two small longitudinal studies found that levels of E-selectin varied little on repeated measurement even in inactive disease, suggesting that low-grade endothelial activation may persist even in the absence of significant clinical disease activity.4 Chronic endothelial activation may contribute to the accelerated atherosclerosis seen in SLE. This could explain the association of E-selectin levels with markers of subclinical atherosclerosis found in this and other studies.15 Rho et al. found that E-selectin and VCAM-1 were significantly associated with coronary artery calcification, independent of other Framingham risk factors while Reynolds et al. recently described an association with persistently raised E-selectin levels and carotid plaque. E-selectin therefore could act as a novel risk marker for atherosclerosis in SLE; however, larger longitudinal studies would be required to investigate this further. The association of E-selectin with SLE-related damaged had been less well studied; however, Rho et al. also found an association with SDI and E-selectin.15 Patients with SLE-related damage are likely to have had more severe or poorly controlled disease, which may lead to chronic endothelial activation as reflected by raised E-selectin levels.In this large cross-sectional cohort, we found raised circulating levels of E-selectin in SLE patients compared with controls and that E-selectin was independently associated both with SLE-related damage and also with carotid plaque. A number of previous studies have also reported raised E-selectin in SLE, but interestingly, few have found a correlation with disease activity.6 In other small studies VCAM-1 levels rose prior to a flare and persistently raised levels following clinical resolution was associated with subsequent relapse.16 The current gold standard for monitoring renal disease activity is serial renal biopsy; however, VCAM-1 could provide an alternative non-invasive monitoring tool. We did not find an association with VCAM-1 and overall disease activity using the SLEDAI-2K. We recognise that two-thirds of patients had low disease activity at the time of assessment and therefore our study may lack the power to detect an association between non-renal inflammation and VCAM-1. Our data do, however, suggest that the association seems specific for active renal disease.Whilst we found no significant difference in VCAM-1 levels in patients and controls, nor an association with disease activity, there was a marked association with active lupus nephritis. Although only five patients had active nephritis, the significant increase in levels was not driven by a single outlier and we observed a genuine trend for higher VCAM-1 levels in all cases with active nephritis see . This as17 In vitro studies have shown some increased expression of VCAM-1 on the surface of endothelial cells following exposure to nicotine.18 The relationship of serum VCAM-1 and smoking status is not yet clear and there may be unmeasured confounding contributing to our result.The negative association with smoking and VCAM-1 was surprising. Smoking is associated with immune activation and it would be expected that smokers may have higher levels of VCAM-1. On review of the literature, however, few studies have found an association with serum VCAM-1 and smoking; in fact, one study found an inverse association in lupus patients.Although this is one of the largest studies evaluating cellular adhesion molecules in SLE patients, the study was not powered to detect associations with clinical CVD. Additionally, the current study population was predominantly Caucasian, hence these results may not be generalisable to other ethnicities.Carotid plaque burden was not measured in the control cohort. As plaque prevalence is significantly lower in the general population, a much larger sample size would have been required to test associations with plaque in the control group and this was not within the scope of this study. As this is a cross-sectional study, we cannot drawn any conclusions about the temporal relationship of CAMs and SLE disease characteristics. Any biomarker should not only be associated with the biological process of interest but must be sensitive to change, thus longitudinal studies are planned to validate the use of these molecules as biomarkers in SLE.Despite these limitations, both E-selectin and VCAM-1 could have utility in clinical practice. E-selectin may help identify patients at increased risk of overall damage and specifically of CVD. VCAM-1 may be a useful biomarker for renal disease activity and supplement current indirect markers of lupus nephritis.


While Brazil has had a long-standing policy of free access to antiretroviral therapy (ART) for all in need, the epidemiological impact of ART on human immunodeficiency virus (HIV) RNA suppression in this middle-income country has not been well evaluated. We estimate first-line ART effectiveness in a large Brazilian cohort and examine the socio-demographic, behavioral, clinical and structural factors associated with virologic suppression.Virologic suppression on first-line ART at 6, 12, and 24 months from start of ART was defined as having a viral load measurement ≤400 copies/mL without drug class modification and/or discontinuation. Drug class modification and/or discontinuation were defined based on the class of a particular drug. Quasi-Poisson regression was used to quantify the association of factors with virologic suppression.From January 2000 through June 2010, 1311 patients started first-line ART; 987 (75%) patients used NNRTI-based regimens. Virologic suppression was achieved by 77%, 76% and 68% of patients at 6, 12 and 24 months, respectively. Factors associated with virologic suppression at 12 months were: >8 years of formal education  1.13, 95% confidence interval (95% CI) 1.03-1.24), starting ART in 2005-2010 , and clinical trial participation . Also at 12 months, women showed less virologic suppression compared to heterosexual men (RR 0.90 95% CI 0.82-0.99). For the 24-month endpoint, in addition to higher education, starting ART in the later period, and clinical trial participation, older age and an NNRTI-based regimen were also independently associated with virologic suppression.Our results show that in Brazil, a middle-income country with free access to treatment, over three-quarters of patients receiving routine care reached virologic suppression on first-line ART by the end of the first year. Higher education, more recent ART initiation and clinical trial participation were associated with improved outcomes both for the 12-month and the 24-month endpoints, suggesting that further studies are needed to understand what aspects relating to these factors lead to higher virologic suppression. Since the beginning of the HIV/AIDS epidemic, Brazil’s response has been both timely and inclusive, addressing prevention as well as treatment. A noteworthy moment was the decision to provide highly active antiretroviral therapy (ART) for all patients in need in 1996. With over 15 years of universal access to ART and almost 300,000 patients receiving ART, Brazil’s HIV treatment program stands alone in its universal coverage of all in need compared to other middle-income or resource-limited countries -4. HowevThe efficacy of new drugs is assessed in short-term randomized clinical trials usually conducted in selected populations which frequently exclude participants with concurrent opportunistic diseases, substance abuse and/or psychiatric comorbidities . As suchThis study was conducted at the Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation (IPEC/FIOCRUZ), one of the largest infectious disease research centers in Brazil, where care has been provided to HIV/AIDS patients since the beginning of the AIDS epidemic in Brazil in 1986. An observational, longitudinal, clinical database is maintained on patients receiving primary and specialized outpatient and inpatient HIV care at the clinic; it includes socio-demographic, behavioral, clinical and therapeutic information. Details of the HIV/AIDS clinical cohort can be found elsewhere ,8. The IAll patients who started first-line ART between January 1, 2000 and June 30, 2010 were included and follow-up information included data through September 30, 2011. Though combination ART became available in mid-1996, we specifically excluded 1996-1999 because it was a period characterized by non-standardized combinations as well as continued exposure to mono or dual therapy, which were kept in the prior to 2000 guidelines as alternative first-line regimens. ART was defined as two nucleoside reverse transcriptase inhibitors (NRTI) in combination with one non-nucleoside reverse transcriptase inhibitor (NNRTI) or one protease inhibitor (PI). Drug class modification and/or discontinuation were defined based on the class of a particular drug. A patient who started a first-line NNRTI-based regimen, for example, was assumed to have modified and/or discontinued the regimen if it was changed to a PI-based regimen or if the NNRTI was discontinued. NRTI modifications and/or discontinuations were not considered since these could be due to reasons unrelated to a drug-class failure. For the years 2000-2010, ART guidelines were consistent regarding first-line ART with NNRTI-based regimens cited as preferred and PI-based regimens as alternative options.First-line ART effectiveness was defined as having HIV viral load ≤ 400 copies/mL and no drug class modification and/or discontinuation. Deaths from AIDS-related causes were considered as failures. Because the limit of detection of viral load assays used throughout the study period varied from ≤400 copies/mL to ≤50 copies/mL, we used the ≤400 copies/mL threshold for the entire study period for consistency.We examined virologic outcomes at 6, 12, and 24 months from first-line ART initiation. Window periods were defined for each time point as 5-9 months, 9-15 months, and 21-27 months, respectively. Within each window, the viral load measurement occurring closest to the target time point (either before or after) was chosen. Drug class modification and/or discontinuations were evaluated for the entire period from the start of first-line ART until the upper limit of each window period.The IPEC Cohort has a validated algorithm for identification of deaths which has been previously described ,9. In adWe examined CD4 counts 6, 12, and 24 months from ART initiation. Window periods were, as for viral loads, defined for each time point as 5-9 months, 9-15 months, and 21-27 months. Within each window, the CD4 count occurring closest to the target time point (either before or after) was chosen.http://www.r-project.org) was used for all statistical analyses.First-line ART effectiveness was calculated as the probability (95% CI) of viral suppression at 6, 12 and 24 months after ART initiation. The impact of socio-demographic, behavioral, clinical and structural factors on virologic suppression at 12 and 24 months was estimated using a quasi-Poisson regression model; this corrected for variance estimation and allowed for the estimation of relative risks. We chose the use of quasi-Poisson models since the data showed overdispersion  and because log-binomial regression models did not converge. The final model included variables found to be significant at a threshold p-value of 0.05, as well as factors known to be clinically relevant or that were shown to modify the effect of a covariate in the adjusted model. We also examined the impact of missing viral load measurements on the final model by re-estimating the parameters while assuming the worst-case and best-case scenarios. R software version 2.15.2  was used by 93.3% (921/987); the most frequent EFV-based combination was zidovudine (AZT) + lamivudine (3TC) + EFV , followed by tenofovir (TDF) + 3TC + EFV or TDF + emtricitabine (FTC) + EFV . Among the 324 patients who started a PI-containing regimen, the majority used a boosted PI . The most frequent boosted PI was ritonavir-boosted lopinavir LPV/r  followed by ritonavir-boosted atazanavir ATV/r . Among those who started a non-boosted PI regimen , 65.4% (83/127) started with ATV. The frequency of boosted PI prescriptions was 47.8% (54/113) for the calendar year 2000-04 and 67.7% (143/211) for 2005-10.Overall first-line ART effectiveness, inclusive only of those with viral load data, was 76.9%, 76.1% and 67.9% at 6, 12 and 24 months . In We showed in a stratified analysis that ART effectiveness was higher for NNRTI-based regimens. These findings corroborate results from clinical trials and cohort studies that demonstrate greater effectiveness of NNRTI-based regimens, in particular, of efavirenz-based regimens ,19,24. IOur study covered a time span of 11 years which allowed us to evaluate ART effectiveness in two periods, namely 2000-2004 and 2005-2010. Both the crude estimates and the adjusted analyses showed that the more recent calendar period was associated with increased ART effectiveness. This finding most likely results from the availability, more recently, of regimens with improved drug combinations, drugs with better tolerability and dosing convenience and, as a result, improved treatment adherence .We found that clinical trial participation was independently associated with virologic suppression at 12 and 24 months from start of ART, corroborating other published results ,30. NotaTwo socio-demographic factors (older age and higher education level) were found to be independently associated with increased ART effectiveness, and there was a trend toward increased virologic suppression for one behavioral factor (MSM HIV risk exposure). Regarding older age, our findings corroborate results from other cohort studies that have found increased ART effectiveness among older individuals ,32. LikeOur study has several limitations. One is the substantial fraction of missing viral load measurements. We addressed this limitation by conducting sensitivity analyses which allowed us to generate upper and lower limits for the ART effectiveness estimates. We also evaluated the impact of the missing viral loads on the adjusted analysis by modeling both best- and worst-case scenarios. These modeling exercises generated results which are similar to those obtained when the missing data were excluded. In contrast, CD4 counts were not imputed and did suffer from a somewhat smaller degree of missing data, and therefore care is needed when extrapolating from these results.In summary, we have shown that in Brazil, a middle-income country universal access to care and treatment, virologic suppression on first-line ART was achieved by over three-quarters of patients receiving routine care in a public facility. We also studied factors associated with virologic suppression at 12- and 24 months since ART initiation and found that higher education, more recent ART initiation and clinical trial participation were associated with improved outcomes. To translate these findings into applicable interventions to improve ART outcomes, the specifics relating to the factors leading to higher virologic suppression need to be further studied.The authors declare no conflict of interest.SWC contributed to the conception and design of the study, acquired the data, and drafted the manuscript. PML contributed to the conception and design of the study, and drafted the manuscript. LV contributed to the conception and design of the study, and performed the statistical analysis. TT and LC acquired and revised the data. KAF participated in the study’s design, discussed the results, helped to draft and revise the manuscript for important intellectual content. VGV participated in the study’s design and coordination and helped to draft the manuscript. RPW participated in the study’s design, discussed the results, helped to draft and revise the manuscript for important intellectual content. BG contributed to the conception and design of the study, coordinated efforts, drafted and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.


Furthermore, matrine inhibited the migration and invasion of NPC tumor cells at doses below the toxic range. Following treatment with matrine for 24 h, there was a decrease in the protein expression levels and activities of matrix metal-loproteinase (MMP)-2 and MMP-9 in NPC-039 cells. In addition, matrine markedly reduced the expression levels of p65 and p50 in the nuclei. Combined treatment of matrine with helenalin, a nuclear factor-κB (NF-κB) inhibitor resulted in a synergistic reduction in MMP-2 and MMP-9 expression levels, and the invasive capabilities of the NPC-039 cells were also reduced. In conclusion, matrine inhibits NPC cell migration and invasion by suppressing the NF-κB pathway. These results suggest that matrine may be a potential therapeutic agent for NPC.Matrine is a widely used Chinese herbal medicine that has historically been used in the treatment of inflammation and cancer. However, the antimetastatic effects and associated molecular mechanisms of matrine on nasopharyngeal carcinoma (NPC) remain to be elucidated. Therefore, the aims of the present study were to assess the antimetastatic effects of matrine on NPC, and identify the underlying mechanisms. Matrine inhibited the proliferation of NPC cells  Nasopharyngeal carcinoma (NPC) is a common malignancy of the head and neck. There is a high incidence of NPC in the southern regions of China, and it is common among the Inuits of Alaska ,2. Due tMetastasis is a complex process, which includes reduction of tumor cell adhesion, degradation of the extracellular matrix (ECM), enhancement of cell motility and promotion of neo-vascularization . Degrada15H24N2O) is derived from the Sophora plant genus and has historically been used in traditional Chinese medicine to treat inflammation  were supplied by the Experimental Animal Center of Xi’an Jiaotong University . The present study was conducted according to the recommended guidelines for the Care and Use of Laboratory Animals, issued by the Chinese Council on Animal Research. The protocol was approved by the Ethics Committee of Xi’an Jiaotong University.Matrine was obtained from Sigma-Aldrich  and was dissolved in dimethyl sulfoxide (DMSO) for cell culture. Fetal bovine serum (FBS), penicillin and streptomycin were all purchased from Gibco Life Technologies . Helenalin was purchased from Sigma-Aldrich. Mouse monoclonal anti-nuclear factor-κ (NF-κB) p50 (sc-271908), mouse monoclonal anti-NF-κ p65 (sc-71676), mouse monoclonal anti-β-actin (sc-376421) and rabbit polyclonal anti-histone H1 (sc-67324) were purchased from Santa Cruz Biotechnology, Inc. . Rabbit polyclonal anti-MMP-2 (#4002) and rabbit polyclonal anti-MMP-9 (#2270) antibodies were purchased from Cell Signaling Technologies .2.The NPC-039 cells were obtained from Academia Sinica  and were was cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS. The poorly differentiated human CNE-2Z NPC cell line was obatined from Zhongshan University  and cultured in RPMI-1640 medium  supplemented with 5% FBS and 100 units penicillin/streptomycin. All of the cells were cultured at 37°C in a humidified incubator containing 5% CO4/well) and treated with serial concentrations  of matrine for 24 h. Following incubation, the cells were washed twice with phosphate-buffered saline (PBS) and incubated with 5 mg/ml MTT (Sigma-Aldrich) for 4 h. The living cells absorbed the reagent and subsequently produced insoluble blue formazan. Following the incubation, the cells were washed again with PBS and solubilized using DMSO. The optical density of the cells was measured at 550 nm using an enzyme-linked immunosorbent assay plate reader .Cell viability was determined using a colorimetric 3- 2, 5-diphenyltetrazolium bromide (MTT) assay, according to previously described methods . Brieflyμm Matrigel™ , and the CNE-2Z cells were pretreated with 0, 12.5, 25 and 50 μg/ml matrine, or helenalin (5 μM), for 24 h. The cells were then seeded in 200 μl serum-free medium and placed in the upper chambers at 2×105 cells, normal growth medium was placed in the bottom chambers. Following a 24 h incubation, the cells on the upper surface of the membrane were removed using cotton swabs, and the cells on the bottom side of the filter were fixed in 90% ethyl alcohol , stained with Crystal Violet (Sigma-Aldrich) and counted under an Olympus-CX31 microscope . Spontaneous migration in DMSO was designated as the control. The rate of invasion was expressed as a percentage of the control.Prior to cell seeding, a 24-well cell culture invasion chamber  was coated with 8.0 μg/ml) of matrine at 37°C for 24 h. The conditioned media were collected from all of the cells and lysated. The protein concentration was then measured and the final 10 μl sample was subjected to electrophoresis using an 8% SDS-PAGE gel co-polymerized with 0.1% gelatin. Following electrophoresis, the gels were incubated in 2.5% Triton X-100 solution (Sigma-Aldrich) at room temperature for 1 h, and then incubated in reaction buffer  overnight at 37°C. The gels were stained with 0.1% Coomassie brilliant blue R-250 (Sigma-Aldrich) and destained with 30% methanol and 10% acetic acid. Gelatinolytic activities were detected as unstained bands against the background of Coomasie-stained gelatin. The intensities of the bands on the gels were determined using an image analysis system .MMP-2 and MMP-9 activity in the samples was studies using zymography. In brief, the cells were treated with different concentrations  or helenalin (5 μM), 1×106 cells were suspended in 200 μl lysis buffer . Nuclear lysates from the cultured NPC-039 cells were harvested using the NucBuster™ Protein Extraction kit , according to the manufacturer’s instructions. The proteins (60 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes . The membranes were subsequently blocked in non-fat milk [5% in Tris-buffered saline with Tween®-20 (TBST) buffer] at 37°C for 1 h, in order to block non-specific binding. The membranes were then incubated overnight with polyclonal antibodies against p50, p65, MMP-2, MMP-9, β-actin and Histone H1, in TBST containing 5% non-fat milk, at 4°C. The membranes were subsequently incubated with a horseradish peroxidase-conjugated goat anti-mouse (EK010) or anti-rabbit (EK020) immunoglobulin G , for 1 h at room temperature. The bands were visualized using an Enhanced Chemiluminescence kit  and exposed by autoradiography . A densitometric analysis was conducted using ImageJ software  and the results are expressed as arbitrary units (a.u.).Following treatment of the NC-039 cells with different concentrations of matrine  were implanted into the right flank region of the BALB/c nude mice. Forty-eight hours after the injection (day 1), the mice were randomly divided into two groups (n=5/group). The animals were pair matched, in order to ensure that the median tumor volume for each group was similar. The treatment group received matrine (60 mg/kg per day) by intragastric administration, and the control group received an equal volume of saline. The tumor volumes were measured twice weekly using calipers, and the volumes (cm3) were calculated according to the following standard formula: (length × width2)/2. After three weeks of drug administration, the mice were sacrificed by cervical dislocation, and the tumors were harvested and weighed. The experimental protocols involving mice in the present study were evaluated and approved by the Animal Care and Use Committee of the Medical School of Xi’an Jiaotong University. methods . BrieflyWound healing assays were performed on NPC-039 cells. In brief, NPC-039 cells were seeded into a six-well plate and cultured to 60–70% confluency in medium containing 10% FBS. Cell monolayers were wounded using a plastic tip (1 mm) that touched the plate as described previously , to evaluate statistical differences. Student’s t-test was used for comparisons between two groups, and a one-way or two-way analysis of variance was used to analyze statistical differences between the groups under different conditions. P<0.05 was considered to indicate a statistically significant difference. All of the statistical tests were two sided. A correlation analysis by Z test was also performed.μg/ml matrine for 24, 48 and 72 h , the antiproliferative effects of matrine were not obvious; therefore, a concentration range lower than this was chosen for use in all subsequent experiments regarding the anti-metastatic effects of matrine.Matrine reduced the viability of the two human NPC cell lines, in a dose-dependent manner, following treatment with 0–200 NPC-039, . At concμg/ml matrine, respectively (μg/ml matrine for 24 h are shown in μg/ml of matrine (As shown in ectively . The eff matrine , respectMMP-2 and MMP-9 have important roles in the invasion of cancer cells . The preNF-κB has an important role in controlling tumor cell migration; therefore, the present study hypothesized that the reduction in NPC cell invasion may be a result of downregulation of NF-κB. The protein expression levels of p65 and p50 in the nuclei of NPC-039 cells were markedly downregulated following treatment with matrine . NF-κB hin vivo effects of matrine on tumor growth, NPC-039 cells were xenografted into nude mice, as described by previous methods (To evaluate the  methods . The tim methods .Metastasis is currently considered to be the main obstacle in the clinical management of NPC. Prevention, prediction and inhibition of NPC metastasis is critical to further improve the survival rate. The present study demonstrated that matrine was a strong metastatic inhibitor for NPC. Matrine has previously been proposed as a potential drug for certain types of tumor ,23. HoweTo determine the effects of matrine on the motility, migration and invasiveness of NPC cells, a wound healing migration assay and a Boyden chamber invasion assay were employed, respectively. Matrine significantly inhibited the migration and invasion of the NPC cells. Metastasis is a complex process, during which degradation of components of the ECM is a key step ; since tNF-κB is able to upregulate MMP-2 and MMP-9 expression –21, and n vivo analyses of the present study demonstrated that matrine decreased the growth of NPC-039 cell tumor xenografts in nude mice by inhibiting cell proliferation. In conclusion, the present study demonstrated the inhibitory effects of matrine on the migration and invasion of NPC cells. Furthermore, the decreased expression levels of MMP-2 and MMP-9, induced by matrine, may be attributed to inhibition of the NF-κB pathway. These results reveal a novel potential therapeutic application of matrine in antimetastatic therapy for NPC.I


Although predator odors are ethologically relevant stimuli for rodents, the molecular pathways and contribution of some brain regions involved in predator odor conditioning remain elusive. Inhibition of histone deacetylases (HDACs) in the dorsal hippocampus has been shown to enhance shock-induced contextual fear learning, but it is unknown if HDACs have differential effects along the dorso-ventral hippocampal axis during predator odor fear learning. We injected MS-275, a class I HDAC inhibitor, bilaterally in the dorsal or ventral hippocampus of mice and found that it had no effects on innate anxiety in either region. We then assessed the effects of MS-275 at different stages of fear learning along the longitudinal hippocampal axis. Animals were injected with MS-275 or vehicle after context pre-exposure (pre-conditioning injections), when a representation of the context is first formed, or after exposure to coyote urine (post-conditioning injections), when the context becomes associated with predator odor. When MS-275 was administered after context pre-exposure, dorsally injected animals showed enhanced fear in the training context but were able to discriminate it from a neutral environment. Conversely, ventrally injected animals did not display enhanced learning in the training context but generalized the fear response to a neutral context. However, when MS-275 was administered after conditioning, there were no differences between the MS-275 and vehicle control groups in either the dorsal or ventral hippocampus. Surprisingly, all groups displayed generalization to a neutral context, suggesting that predator odor exposure followed by a mild stressor such as restraint leads to fear generalization. These results may elucidate distinct functions of the dorsal and ventral hippocampus in predator odor-induced fear conditioning as well as some of the molecular mechanisms underlying fear generalization. Predator odors are ethologically relevant stimuli that have been shown to elicit a variety of defensive responses in rodents  were housed individually, kept on a 12-h light/dark cycle, and allowed access to food and water Animals were run in the open-field test and black-white box to determine the effects of MS-275 on innate anxiety. MS-275 was infused bilaterally into either the dorsal or ventral hippocampus 1–2 h before conducting anxiety tests. For the open field test, mice were placed in the center of a large cylindrical arena (70 cm in diameter). The arena was illuminated by a ceiling-mounted array of eight 60-watt lights arranged symmetrically around the perimeter, approximately 1.8 m above the base. Explorative behavior was recorded for 20 min. Additionally, we evaluated freezing by calculating the percent time the animals remain immobile, except for respiratory movements. For the black/white two compartment box, we used a plastic box divided into two equal compartments (22 × 24 cm) connected by a small opening. The black compartment was darkened with black contact paper and covered with a piece of cardboard, while the open-topped white compartment was lined with white contact paper and illuminated by three ceiling-mounted 60-watt lights approximately 1 m above the apparatus, aimed at the center of the compartment. Mice were placed in the center of the white compartment facing the black side, and explorative behavior was recorded for 3 min .Prior to the start of behavioral experiments, animals were handled and restrained twice a day for 2 consecutive days. Animals were then conditioned using a predator odor contextual fear conditioning paradigm previously characterized in our lab . For the open-field test, the context was divided into three equally spaced concentric circles and the percent time spent in these areas was measured. For the black-white box, the percent time spent in the white compartment of the white/black box was measured, along with the number of reentries to the white side. For conditioning and the open field, freezing was quantified as the percentage of time during which the velocity of the animal was lower than 0.6 cm/s. Freezing was evaluated using both Lime Light and custom-written MATLAB code. For the MATLAB analysis, the position data were smoothed with a 1 s boxcar to eliminate jitter in the tracking. Finally, we calculated percent freezing by calculating the percent ratio between freezing at 24 h and freezing during baseline for each context.Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and placed on a stereotaxic apparatus in a flat skull position . 26 gauge guide cannulas  were implanted bilaterally in either the dorsal or ventral hippocampus at the following coordinates, measured from Bregma in mm. Dorsal: AP, -1.7; ML, ± 1.5; DV: -1.0 . Ventral: AP, -3.0; ML, ± 2.8; DV: -2.0 . An anchor screw was placed just anterior to lambda, and cannulas were affixed to the skull with cyanoacrylate and dental cement. After surgery, animals were allowed to recover for 1 week prior to behavioral experiments.3, with each hemisphere being approximately 14 mm3 and each dorsal and ventral sub-regions around 7 mm3  was diluted to 1 mM using 4% dimethyl sulfoxide (DMSO) in ACSF, then infused bilaterally into either the dorsal or ventral hippocampus through the implanted guide cannulas using a standard infusion syringe pump . Total volume injected was 0.5 μl on each side at a rate of 0.5 μl/min. Controls were injected with an equivalent volume of vehicle . We estimated the concentration of MS-275 in the hippocampus to be roughly 71 μM, since the average volume of the hippocampus in C57bl6 mice is 28 mmTo verify cannula placements, animals were sacrificed after behavioral experiments. Brains were removed and fixed at 4°C with 10% formalin for at least 24 h. They were then transferred to a 30% sucrose solution and kept for at least 48 h at 4°C for cryoprotection. Brains were then cryosectioned  and Nissl stained with cresyl violet using standard histological procedures , and 41 animals were injected with vehicle . We divided the open field in three concentric areas and calculated the time spent in each of these zones. We did not find any differences between the groups in the percentage of time spent in the three concentric regions comprising the open field . Additionally, we did not find differences in the levels of freezing in the open field .Since we wanted to establish the effects of HDAC inhibition on fear learning, we first investigated whether MS-275 had any effects on innate anxiety. We performed bilateral injections of MS-275 or vehicle into the dorsal or ventral hippocampus of animals 2–3 h prior to behavioral testing in the open field and the black/white box Figures . The opeN = 12; ventral: N = 11) and 23 animals were injected with vehicle . We did not find significant differences between the groups in the percentage of time spent in the white compartment  or the number of reentries to the white compartment . These results suggest that MS-275 does not affect innate anxiety in the dorsal or ventral hippocampus.Next, we evaluated the effects of MS-275 on behavior in a black/white two-compartment box  was injected in either the dorsal or ventral hippocampus after exposure to the training context (baseline context A) and a neutral context (baseline context B) on Day 1 (pre-conditioning injections). Twenty-four hours after the injections (Day 2), these groups were conditioned and tested 1 h after conditioning in the training context A. On day 3, the four groups were retested in the training (A) and neutral (B) context 24 h after conditioning . In support of our hypothesis, we found that dorsal injections significantly increased freezing in the training context (context A) in the MS-275 group in comparison to the vehicle control group.  = 4.50, p < 0.05, effect of session: F = 17.56, p < 0.001; interaction: F = 2.95, p < 0.04). Post hoc Student-Newman-Keuls tests (SNKTs) showed that freezing levels were comparable in the control and MS-275 groups during baseline , but were significantly different during the post-conditioning 24 h test in context A (24 h: p < 0.003). Furthermore, although MS-275 also produced a significant increase in freezing in the neutral context (B) (SNKTs: 24 h B: p < 0.03), both the vehicle and MS-275 groups, were able to discriminate the training context A from the neutral context B, which was evident in significantly higher levels of freezing in context A . The differences between the MS-275 and vehicle groups are also evident in a significant percent increase in freezing observed 24 h after conditioning relative to baseline in context A  and a trend in context B . Again, when we compared the percent increase in freezing from baseline within each group, both the MS-275 and vehicle control groups displayed higher freezing in context A than B at 24 h relative to the freezing baseline in each context (MS-275 (trend toward significance): t11 = 2.13, p = 0.056; vehicle control: t19 = 2.63, p < 0.02). These results suggest that MS-275 enhances the memory of the training context without disrupting the ability of the animals to discriminate this context from a neutral one.There were no differences in baseline freezing prior to conditioning in the dorsal groups . Even though MS-275 did not significantly affect learning, the HDAC inhibitor produced fear generalization in response to the neutral context B. This was evident in higher freezing levels in the neutral context in animals injected with MS-275 than vehicle-injected controls  = 0.09, p = 0.77, effect of session: F = 25.19, p < 0.001; interaction: F = 2.75, p < 0.05). SNKTs showed no differences prior to conditioning or after conditioning in the training context (P > 0.05). However, the groups were significantly different in the neutral context B (24 h B: p < 0.04). Furthermore, while the control animals discriminated between the training and neutral context (p < 0.003), the MS-275 group did not (p = 0.865). These effects are also evident when we examined the percent increase in freezing at 24 h after conditioning relative to baseline in each context, showing that there were no differences in the training context A  but significantly higher freezing in the MS-275 group in context B , MS-275 animals showed no difference in freezing in context A and B . These results suggest that HDAC I inhibition in the dorsal and ventral hippocampus plays different roles in predator odor fear memory. MS-275 leads to enhanced fear memory in the dorsal hippocampus and fear generalization in the ventral region.In the ventral hippocampus, we did not observe differences in baseline freezing between the groups receiving injections after contextual pre-exposure  after conditioning  = 0.25, p = 0.62, interaction: F = 0.06, p = 0.98). The MS-275 (N = 11) and vehicle control (N = 11) groups only showed an effect of session reflecting that freezing was significantly higher after learning  = 17.04, p < 0.001; post hoc comparisons showed that freezing was significantly higher after conditioning in comparison to baseline in both context A and B, p < 0.05). In the ventral groups, we observed a similar pattern. There were also no significant differences between the MS-275 (N = 13) and control (N = 14) groups  = 0.51, p = 0.48, interaction: F = 1.13, p = 0.34), though the groups displayed a significant effect of session, which reflected that freezing changed after learning  = 27.19, p < 0.001, post hoc comparisons showed that freezing was significantly higher after conditioning in comparison to baseline in both context A and B, p < 0.05). The fact that all groups exhibited significantly higher freezing in the neutral context B at 24 h compared to baseline indicates that generalization is not produced by HDAC inhibition. Since all animals are restrained during the injections, we suggest that the observed generalization occurs as a result of having an additional stressor following predator odor exposure. These findings indicate that predator odor fear learning provides a good model to study fear generalization.In agreement with our predictions, we found no significant effect of MS-275 in the dorsal hippocampus  has different effects in the dorsal and ventral hippocampus. In the dorsal hippocampus, HDAC inhibition enhances fear learning, whereas in the ventral hippocampus, it leads to fear generalization. However, this epigenetic mechanism does not affect learning when HDAC inhibition takes place after conditioning. Instead, the presentation of a predator odor followed by a stressor results in fear generalization to a neutral context.There are several advantages associated with the use of predator odors in fear learning. First, odors are extremely relevant cues to rodents because identifying dangerous odors is critical for survival  display increased freezing in a neutral context, the level of generalization observed in these animals is minimal in comparison to ventrally injected mice. This suggests that these animals still differentiate between the training and neutral contexts. Conversely, animals injected with MS-275 in the ventral region cannot discriminate between neutral and fearful contexts. Several studies support the idea that the ventral hippocampus may play a role in fear generalization. In rats, hippocampal place cells have receptive fields of increasing size moving from the dorsal to the ventral pole  CAREER award to IM (grant number 1256941) and a Ruth L. Kirschstein predoctoral National Research Service Award (NRSA) to RY (grant number 1F31MH105161-01A1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.